Project III: Latent metastasis: Immune Regulation Of Disseminated Cancer Stem Cells PROJECT SUMMARY: The goal of Project III is to discover mechanisms that critically regulate immune evasion by disseminated tumor cells (DTCs) and their evolution as latent metastatic entities. We will use an integrated approach that combines single-cell interrogation methods with unique biological models of latent metastasis from breast cancer and lung adenocarcinoma, and novel computational strategies. Distant metastasis underlies the overwhelming majority of cancer-related deaths and its inception is exceedingly variable. Residual DTCs may outgrow immediately or, more frequently, linger in a viable state of replicative quiescence or mass dormancy for months to years after infiltrating distant organs. This latency state of DTCs is accompanied with significant resistance to anti-neoplastic therapy, which typically targets actively dividing tumor cells. Moreover, latent DTCs somehow evade immune surveillance. The biology underlying these adaptive abilities remains poorly understood and factors governing DTC population dynamics, whether stochastic or deterministic, remain unknown. We aim to address this significant knowledge gap by combining massively parallel, single-cell RNA expression profiling using a bead-based molecular barcoding technology with unsupervised learning methods to identify stable/transitory cell states within latent, residual disease and their molecular control mechanisms. As a complementary approach, individual cell responses to molecular perturbations will be dynamically tracked by live cell imaging. In Aim 1 we propose to determine whether the latent state pre-exists in the primary tumor or is induced by the stress of immunosurveillance in a host tissue. In Aim 2 we will model the evolutionary dynamics of metastatic cells as they exit latency under growth permissive and immunoediting conditions. Moreover, in Aim 3 we will identify key regulators of metastatic immune evasion by probing transcriptional heterogeneity in quiescent subpopulations differentially sensitive to NK-cell mediated elimination. The amalgamation of these approaches, combined with our deep understanding of the biology of cancer metastasis, will promote the discovery of therapeutic strategies to eradicate or control metastasis from its earliest stages of inception.